Libraries

library(tidyverse)

## ── Attaching packages ────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────── tidyverse 1.2.1 ──

## ✓ ggplot2 3.3.0     ✓ purrr   0.3.3
## ✓ tibble  3.0.1     ✓ dplyr   0.8.5
## ✓ tidyr   1.0.0     ✓ stringr 1.4.0
## ✓ readr   1.3.1     ✓ forcats 0.4.0

## Warning: package 'tibble' was built under R version 3.6.2

## ── Conflicts ───────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────── tidyverse_conflicts() ──
## x dplyr::filter() masks stats::filter()
## x dplyr::lag()    masks stats::lag()

library(janitor)

## 
## Attaching package: 'janitor'

## The following objects are masked from 'package:stats':
## 
##     chisq.test, fisher.test

library(knitr)
library(cowplot) # for plot_grid and draw_label

## 
## ********************************************************

## Note: As of version 1.0.0, cowplot does not change the

##   default ggplot2 theme anymore. To recover the previous

##   behavior, execute:
##   theme_set(theme_cowplot())

## ********************************************************

library(RColorBrewer)
library(pander) # to make session info
library(kableExtra)

## 
## Attaching package: 'kableExtra'

## The following object is masked from 'package:dplyr':
## 
##     group_rows

library(snakecase)
library(corrr)

Config

inputs_dir <- "inputs"
file_pattern <- "survey_data"
data_file <- tibble(files = list.files(inputs_dir, file_pattern)) %>%
  arrange(desc(files)) %>%
  top_n(1, wt = files) %>%
  pull(files)

print(data_file)

## [1] "survey_data.2020-07-29_11.35.40.txt"

Settings

min_genes_gt_0 <- 100

Functions

is_outlier <- function(x) {
  (x > quantile(x, 0.75) + 1.5*IQR(x)) |
    (x < quantile(x, 0.25) - 1.5*IQR(x))
}


most_common_val_w_percent <- function(value_vector = c("a", "a", "b")){
  mc_vals <- tabyl(value_vector) %>%
    arrange(desc(n)) %>%
    top_n(1, n) 
  paste0(round(100*mc_vals$percent), "% ", mc_vals$value_vector)
}

# StatBin2 allows depiction of empty bins as blank instead of a horizontal line:
# https://stackoverflow.com/questions/57128090/remove-baseline-color-for-geom-histogram
StatBin2 <- ggproto(
  "StatBin2", 
  StatBin,
  compute_group = function (data, scales, binwidth = NULL, bins = NULL, 
                            center = NULL, boundary = NULL, 
                            closed = c("right", "left"), pad = FALSE, 
                            breaks = NULL, origin = NULL, right = NULL, 
                            drop = NULL, width = NULL) {
    if (!is.null(breaks)) {
      if (!scales$x$is_discrete()) {
        breaks <- scales$x$transform(breaks)
      }
      bins <- ggplot2:::bin_breaks(breaks, closed)
    }
    else if (!is.null(binwidth)) {
      if (is.function(binwidth)) {
        binwidth <- binwidth(data$x)
      }
      bins <- ggplot2:::bin_breaks_width(scales$x$dimension(), binwidth, 
                                         center = center, boundary = boundary, 
                                         closed = closed)
    }
    else {
      bins <- ggplot2:::bin_breaks_bins(scales$x$dimension(), bins, 
                                        center = center, boundary = boundary, 
                                        closed = closed)
    }
    res <- ggplot2:::bin_vector(data$x, bins, weight = data$weight, pad = pad)

    # drop 0-count bins completely before returning the dataframe
    res <- res[res$count > 0, ] 

    res
  })

Import data

col_spec <- cols(
  .default = col_double(),
  dataset_id = col_character(),
  disease = col_character(),
  pedaya = col_character(),
  race = col_character(),
  ethnicity = col_character(),
  gender = col_character(),
  cohort_name = col_character(),
  cohort_code = col_character(),
  doi_link = col_character(),
  source_repository = col_character(),
  repository_cohort_accession = col_character(),
  repository_dataset_accession = col_character()
)

counts_and_meta_raw <- read_tsv(file.path(inputs_dir, data_file), col_types = col_spec)

counts_and_meta <- counts_and_meta_raw %>%
  group_by(cohort_code) %>%
  mutate(n_in_cohort = n())

write_csv(counts_and_meta, "results/Table_S1.csv")

Colors

# brewer.pal(8, "Paired")

these_colors <- c("#A6CEE3", "#1F78B4", "#B2DF8A", "#33A02C", "#FB9A99", "#E31A1C", 
"#FDBF6F", "#FF7F00")

# light blue, dark blue, etc: green, red, orange (then purple and brown)

these_colors <- c("#1F78B4", "#1F78B4", "#33A02C", "#33A02C", "#E31A1C", "#E31A1C", 
"#FF7F00", "#FF7F00")

names(these_colors) <-  c("not assigned", "Total reads", "Non-exonic reads",  "Exonic reads", "Duplicate reads",  "Non-duplicate reads",  "Unmapped reads", "Mapped reads")

these_colors_with_MEND = c(these_colors, "MEND reads"="#000000")

Datasets analyzed

nrow(counts_and_meta)

## [1] 2179

Diseases in dataset

counts_and_meta %>% tabyl(disease)

##                                     disease   n      percent
##                acute lymphoblastic leukemia 680 0.3120697568
##             acute megakaryoblastic leukemia 103 0.0472693896
##                      acute myeloid leukemia 221 0.1014226709
##             acute undifferentiated leukemia   1 0.0004589261
##                      adrenocortical adenoma   1 0.0004589261
##                       adrenocortical cancer   2 0.0009178522
##                    adrenocortical carcinoma  20 0.0091785223
##                   alveolar rhabdomyosarcoma  40 0.0183570445
##                          cholangiocarcinoma   1 0.0004589261
##                    choroid plexus carcinoma  25 0.0114731528
##         desmoplastic small round cell tumor   4 0.0018357045
##      dysembryoplastic neuroepithelial tumor  13 0.0059660395
##                  embryonal rhabdomyosarcoma  42 0.0192748967
##  embryonal tumor with multilayered rosettes   1 0.0004589261
##                                  ependymoma  98 0.0449747591
##                         epithelioid sarcoma   1 0.0004589261
##                               Ewing sarcoma  70 0.0321248279
##      fibrolamellar hepatocellular carcinoma   3 0.0013767783
##                                fibromatosis   1 0.0004589261
##              gastrointestinal stromal tumor   4 0.0018357045
##                             germ cell tumor   1 0.0004589261
##                     glioblastoma multiforme  29 0.0133088573
##                                      glioma 193 0.0885727398
##                              hepatoblastoma   1 0.0004589261
##                    hepatocellular carcinoma   3 0.0013767783
##                 hypereosinophillic syndrome   1 0.0004589261
##            juvenile myelomonocytic leukemia   1 0.0004589261
##                                    leukemia   1 0.0004589261
##                                    lymphoma  49 0.0224873795
##                             medulloblastoma 201 0.0922441487
##                                    melanoma   7 0.0032124828
##             melanotic neuroectodermal tumor   1 0.0004589261
##                                  meningioma   1 0.0004589261
##                             myofibromatosis   2 0.0009178522
##                    nasopharyngeal carcinoma   2 0.0009178522
##                               neuroblastoma  12 0.0055071134
##                    neuroendocrine carcinoma   1 0.0004589261
##                                not reported   2 0.0009178522
##                not reported - QC fail by PI   1 0.0004589261
##                                osteosarcoma 157 0.0720513997
##           ovarian serous cystadenocarcinoma   1 0.0004589261
##                                      PEComa   1 0.0004589261
##                    pleuropulmonary blastoma   1 0.0004589261
##                              retinoblastoma   2 0.0009178522
##                              rhabdoid tumor  65 0.0298301973
##                            rhabdomyosarcoma  53 0.0243230840
##    spindle cell/sclerosing rhabdomyosarcoma   3 0.0013767783
##          supratentorial embryonal tumor NOS  18 0.0082606700
##                            synovial sarcoma  22 0.0100963745
##                undifferentiated sarcoma NOS   3 0.0013767783
##       undifferentiated spindle cell sarcoma   2 0.0009178522
##                                 wilms tumor  11 0.0050481872

disease_freq_table <- counts_and_meta$disease %>%
  str_to_sentence %>%
  str_replace(" nos$", " NOS") %>%
  as.factor %>%
  fct_infreq %>%
  fct_lump(prop=0.01) %>%
  tabyl (var1 = "Disease") %>%
  adorn_pct_formatting(digits = 1)

colnames(disease_freq_table)[1]="Disease"

write_tsv(disease_freq_table, "results/disease_freq_table.tsv")
write_csv(disease_freq_table, "results/disease_freq_table.csv")

disease_freq_table  %>%
  kable %>% 
  kable_styling(bootstrap_options = "striped", full_width = F)

Disease	n	percent
Acute lymphoblastic leukemia	680	31.2%
Acute myeloid leukemia	221	10.1%
Medulloblastoma	201	9.2%
Glioma	193	8.9%
Osteosarcoma	157	7.2%
Acute megakaryoblastic leukemia	103	4.7%
Ependymoma	98	4.5%
Ewing sarcoma	70	3.2%
Rhabdoid tumor	65	3.0%
Rhabdomyosarcoma	53	2.4%
Lymphoma	49	2.2%
Embryonal rhabdomyosarcoma	42	1.9%
Alveolar rhabdomyosarcoma	40	1.8%
Glioblastoma multiforme	29	1.3%
Choroid plexus carcinoma	25	1.1%
Synovial sarcoma	22	1.0%
Other	131	6.0%

# many are leukemias
table(str_detect(counts_and_meta$disease, "leukemia"))

## 
## FALSE  TRUE 
##  1172  1007

Cohorts in dataset

old_cohort_size_histogram <- ggplot(counts_and_meta) + 
  geom_histogram(aes(n_in_cohort, group=cohort_code), 
                 fill = "lightgrey", color = "black", stat = StatBin2) +
  theme_minimal(base_size = 20) +
  theme(axis.line.x = element_line(color="darkgrey", size = 0.5),
        axis.line.y = element_line(color="darkgrey", size = 0.5)) +  xlab("Cohort size") +
  ylab("Cohorts") + 
  theme(legend.position="none")


# max(table(counts_and_meta$n_in_cohort)/as.numeric(names(table(counts_and_meta$n_in_cohort))))
cohort_hist_bin_size <- 10


cohort_size_histogram_raw <- ggplot(counts_and_meta %>%
  select(cohort_code, n_in_cohort) %>%
  distinct ) + 
  geom_histogram(aes(n_in_cohort), 
                 fill = "lightgrey", color = "black", stat = StatBin2,
                 breaks = seq(0,500, by=10)) +
  theme_minimal(base_size = 20) +
  theme(axis.line.x = element_line(color="darkgrey", size = 0.5),
        axis.line.y = element_line(color="darkgrey", size = 0.5)) +  
  xlab("Cohort size") +
  theme(legend.position="none")

max_y_val <- max(ggplot_build(cohort_size_histogram_raw)$data[[1]][,"y"])


cohort_size_histogram <- cohort_size_histogram_raw + 
  scale_y_continuous("Cohorts", breaks = seq(0, ceiling(max_y_val/2) * 2, 2)) 
  

cohort_size_histogram

n_distinct(counts_and_meta$cohort_code)

## [1] 48

sort(unique(counts_and_meta$n_in_cohort))

##  [1]   3   4   6   7   8  10  15  17  19  22  23  24  25  26  31  35  39
## [18]  43  44  54  56  62  63  65  78  82  84  88  89 103 127 137 141 337

counts_and_meta %>%
  select(cohort_code, n_in_cohort) %>%
  distinct %>%
  arrange(desc(n_in_cohort)) %>%
  kable

cohort_code	n_in_cohort
Cohort_1	337
Cohort_2	141
Cohort_3	137
Cohort_4	127
Cohort_5	103
Cohort_6	89
Cohort_7	88
Cohort_8	84
Cohort_9	82
Cohort_10	78
Cohort_11	65
Cohort_12	63
Cohort_13	62
Cohort_14	56
Cohort_15	54
Cohort_16	44
Cohort_17	43
Cohort_18	39
Cohort_19	35
Cohort_20	31
Cohort_21	31
Cohort_22	26
Cohort_23	25
Cohort_24	25
Cohort_26	24
Cohort_25	24
Cohort_30	23
Cohort_27	23
Cohort_28	23
Cohort_29	23
Cohort_31	22
Cohort_32	19
Cohort_33	19
Cohort_34	17
Cohort_35	15
Cohort_36	10
Cohort_37	8
Cohort_38	8
Cohort_41	7
Cohort_39	7
Cohort_40	7
Cohort_42	6
Cohort_44	6
Cohort_43	6
Cohort_45	6
Cohort_47	4
Cohort_46	4
Cohort_48	3

counts_and_meta %>%
  select(cohort_code, n_in_cohort) %>%
  distinct %>%
  pull(n_in_cohort) %>%
  median

## [1] 24.5

Table S2 - repositories

repo_info <- read_tsv("inputs/repos.tsv")

## Parsed with column specification:
## cols(
##   repo_name = col_character(),
##   repo_abbrev = col_character(),
##   repo_url = col_character(),
##   source_accession_pattern = col_character()
## )

repo_table <- repo_info %>%
  filter(repo_abbrev %in% counts_and_meta$source_repository) %>%
  rename(`Name`=repo_name, `Abbreviation`=repo_abbrev, URL = repo_url) %>%
  select(-source_accession_pattern) 

write_tsv(repo_table, "results/Table_S2_repo_table.tsv")
write_csv(repo_table, "results/Table_S2_repo_table.csv")

repo_table %>%
  kable %>% 
  kable_styling(bootstrap_options = "striped", full_width = F)

Name	Abbreviation	URL
Short Read Archive	SRA	https://www.ncbi.nlm.nih.gov/sra
European Genome-phenome Archive	EGA	https://www.ebi.ac.uk/ega/home
St. Jude Cloud	SJC	https://www.stjude.cloud/
Database of Genotypes and Phenotypes	dbGaP	https://www.ncbi.nlm.nih.gov/gap/
Cavatica	Cavatica	https://cavatica.squarespace.com/

Table S3 - data access table

cohort_table <- counts_and_meta %>% 
  select(cohort_code, cohort_name, source_repository, repository_cohort_accession, n_in_cohort) %>% # removed DOI link
#  group_by(across(c(-disease)))
  distinct %>%
  arrange(desc(n_in_cohort)) 

colnames(cohort_table) <- to_any_case(colnames(cohort_table), case = "sentence")

write_tsv(cohort_table, "results/Table_S3_cohort_table.tsv")
write_csv(cohort_table, "results/Table_S3_cohort_table.csv")




cohort_table %>% 
  kable %>% 
  kable_styling(bootstrap_options = "striped", full_width = F)

Cohort code	Cohort name	Source repository	Repository cohort accession	N in cohort
Cohort_1	TARGET-10	dbGaP	phs000218	337
Cohort_2	SJC_BALL	SJC	SJC-DS-1001	141
Cohort_3	TARGET-20	dbGaP	phs000218	137
Cohort_4	EGAD00001003279	EGA	EGAD00001003279	127
Cohort_5	SJC_AMLM	SJC	SJC-DS-1001	103
Cohort_6	phs000673	dbGaP	phs000673	89
Cohort_7	TARGET-40	dbGaP	phs000218	88
Cohort_8	phs000720	dbGaP	phs000720	84
Cohort_9	SJC_HGG	SJC	SJC-DS-1001	82
Cohort_10	SJC_EPD	SJC	SJC-DS-1001	78
Cohort_11	TARGET-52	dbGaP	phs000218	65
Cohort_12	EGAD00001001098	EGA	EGAD00001001098	63
Cohort_13	phs000768	dbGaP	phs000768	62
Cohort_14	SJC_ETV	SJC	SJC-DS-1001	56
Cohort_15	SJC_LGG	SJC	SJC-DS-1001	54
Cohort_16	SJC_CBF	SJC	SJC-DS-1001	44
Cohort_17	SJC_RHB	SJC	SJC-DS-1001	43
Cohort_18	EGAD00001001620	EGA	EGAD00001001620	39
Cohort_19	phs000699	dbGaP	phs000699	35
Cohort_20	CBTTC	Cavatica	CBTTC	31
Cohort_21	SJC_ERG	SJC	SJC-DS-1001	31
Cohort_22	SJC_PHALL	SJC	SJC-DS-1001	26
Cohort_23	EGAD00001001666	EGA	EGAD00001001666	25
Cohort_24	SJC_CPC	SJC	SJC-DS-1001	25
Cohort_26	phs000900	dbGaP	phs000900	24
Cohort_25	EGAD00001000648	EGA	EGAD00001000648	24
Cohort_30	TARGET-21	dbGaP	phs000218	23
Cohort_27	EGAD00001000356	EGA	EGAD00001000356	23
Cohort_28	EGAD00001000617	EGA	EGAD00001000617	23
Cohort_29	SJC_OS	SJC	SJC-DS-1001	23
Cohort_31	EGAD00001001927	EGA	EGAD00001001927	22
Cohort_32	EGAD00001002680	EGA	EGAD00001002680	19
Cohort_33	SRP126664	SRA	SRP126664	19
Cohort_34	EGAD00001000158	EGA	EGAD00001000158	17
Cohort_35	SRP092501	SRA	SRP092501	15
Cohort_36	EGAD00001000826	EGA	EGAD00001000826	10
Cohort_37	SJC_E	SJC	SJC-DS-1001	8
Cohort_38	SJC_MB	SJC	SJC-DS-1001	8
Cohort_41	TARGET-30	dbGaP	phs000218	7
Cohort_39	SJC_HYPO	SJC	SJC-DS-1001	7
Cohort_40	SJC_MEL	SJC	SJC-DS-1001	7
Cohort_42	EGAD00001000328	EGA	EGAD00001000328	6
Cohort_44	SJC_Other	SJC	SJC-DS-1001	6
Cohort_43	SJC_INF	SJC	SJC-DS-1001	6
Cohort_45	SJC_WLM	SJC	SJC-DS-1001	6
Cohort_47	TARGET-50	dbGaP	phs000218	4
Cohort_46	SRP006575	SRA	SRP006575	4
Cohort_48	SRP040454	SRA	SRP040454	3

Race and gender in dataset

counts_and_meta %>%
  tabyl(race)

##                                       race    n      percent valid_percent
##                                      Asian   27 0.0123910050   0.044850498
##                     Black/African American   70 0.0321248279   0.116279070
##  Native Hawaiian or Other Pacific Islander    3 0.0013767783   0.004983389
##         not-white (no other info provided)    1 0.0004589261   0.001661130
##                                      Other    7 0.0032124828   0.011627907
##                                      White  494 0.2267094998   0.820598007
##                                       <NA> 1577 0.7237264800            NA

counts_and_meta %>%
  select(race) %>%
  na.omit() %>%
  tabyl(race) %>%
  adorn_totals("row")

## Adding missing grouping variables: `cohort_code`

##                                       race   n     percent
##                                      Asian  27 0.044850498
##                     Black/African American  70 0.116279070
##  Native Hawaiian or Other Pacific Islander   3 0.004983389
##         not-white (no other info provided)   1 0.001661130
##                                      Other   7 0.011627907
##                                      White 494 0.820598007
##                                      Total 602 1.000000000

counts_and_meta %>%
    select(ethnicity) %>%
  na.omit() %>%
tabyl(ethnicity) %>%
  adorn_totals("row")

## Adding missing grouping variables: `cohort_code`

##               ethnicity   n   percent
##      Hispanic or Latino 128 0.1486643
##  Not Hispanic or Latino 733 0.8513357
##                   Total 861 1.0000000

counts_and_meta %>%
  filter(ethnicity == "Hispanic or Latino") %>%
  tabyl(race)

##                    race  n   percent valid_percent
##  Black/African American  1 0.0078125    0.01123596
##                   Other  3 0.0234375    0.03370787
##                   White 85 0.6640625    0.95505618
##                    <NA> 39 0.3046875            NA

counts_and_meta %>%
  filter(ethnicity == "Hispanic or Latino") %>%
  select(race) %>%
  na.omit() %>%
tabyl(race) %>%
  adorn_totals("row")

## Adding missing grouping variables: `cohort_code`

##                    race  n    percent
##  Black/African American  1 0.01123596
##                   Other  3 0.03370787
##                   White 85 0.95505618
##                   Total 89 1.00000000

Genders in dataset

table(counts_and_meta$gender, useNA = "always")

## 
## female   male   <NA> 
##    709    983    487

# samples with reported genders
counts_and_meta %>% 
  filter(! is.na(gender)) %>%
  filter(! gender %in% c("not reported", "unknown")) %>%
  tabyl(gender) %>%
  adorn_totals

##  gender    n   percent
##  female  709 0.4190307
##    male  983 0.5809693
##   Total 1692 1.0000000

Ages in dataset

table(counts_and_meta$pedaya, useNA = "always")

## 
##                  No Yes, age < 30 years                <NA> 
##                  67                2088                  24

# at least one target sample is >30

n_ped_aya <- sum(counts_and_meta$pedaya=="Yes, age < 30 years" | counts_and_meta$age_at_dx<30, na.rm = TRUE)
n_ped_aya

## [1] 2088

n_ped_aya/nrow(counts_and_meta)

## [1] 0.9582377

# more than 95% of which are pediatric <30; of the Datasets with specific ages at diagnosis reported, the median was 7

median(counts_and_meta$age_at_dx, na.rm = TRUE)

## [1] 8.32

table(is.na(counts_and_meta$age_at_dx))

## 
## FALSE  TRUE 
##  1472   707

Read lengths in dataset

seq_len_round_25 <- round(counts_and_meta$seq_length / 25) * 25
table(seq_len_round_25, useNA = "always")

## seq_len_round_25
##   25   50   75  100  125 <NA> 
##    4  238  366 1544   27    0

tabyl(seq_len_round_25)

##  seq_len_round_25    n     percent
##                25    4 0.001835704
##                50  238 0.109224415
##                75  366 0.167966957
##               100 1544 0.708581918
##               125   27 0.012391005

table(is.na(counts_and_meta$seq_length))

## 
## FALSE 
##  2179

table(counts_and_meta$seq_length)

## 
##   36   50   51   75   76   81  100  101  110  125 
##    4   11  227  279   40   47   98 1431   15   27

summary(counts_and_meta$seq_length)

##    Min. 1st Qu.  Median    Mean 3rd Qu.    Max. 
##   36.00   76.00  101.00   91.51  101.00  125.00

IQR(counts_and_meta$seq_length, na.rm = TRUE)

## [1] 25

 old_read_length_histogram <-  ggplot(counts_and_meta) + 
  geom_histogram(aes(seq_length, group=cohort_code), 
                 fill = "lightgrey", color = "black", stat = StatBin2) +
      theme_minimal(base_size = 20) +
    theme(axis.line.x = element_line(color="darkgrey", size = 0.5),
        axis.line.y = element_line(color="darkgrey", size = 0.5)) +
  xlab("Sequence length") +
    ylab("Datasets")
 
 read_length_histogram <-  ggplot(counts_and_meta) + 
  geom_histogram(aes(seq_length), 
                 fill = "lightgrey", color = "black", stat = StatBin2,
                 binwidth = 1) +
      theme_minimal(base_size = 20) +
    theme(axis.line.x = element_line(color="darkgrey", size = 0.5),
        axis.line.y = element_line(color="darkgrey", size = 0.5)) +
  xlab("Sequence length") +
    ylab("Datasets") +
   expand_limits(x=0)
  
read_length_histogram

# Figure 1

figure_name <- "f1"



f1 <- plot_grid(cohort_size_histogram,
read_length_histogram,
nrow=2,
labels = c("A", "B")) +
   draw_label(paste(figure_name, Sys.time()),
              x = 0, y = 0.02, hjust = 0, size = 6) 

f1

ggsave("results/Figure_1.png", f1, height=6, width=5)
ggsave("results/Figure_1.pdf", f1, height=6, width=5)

Check for counting errors

if(nrow(subset(counts_and_meta, Mapped > Total_reads)) > 0) stop("Counting error")
if(nrow(subset(counts_and_meta, MND > Mapped)) > 0) stop("Counting error")
if(nrow(subset(counts_and_meta, MEND > MND)) > 0) stop("Counting error")

Summarize total read counts

min(counts_and_meta$Total_reads)

## [1] 206916

Total_read_counts <- counts_and_meta %>% 
  ungroup %>%
  mutate(dataset_value = Total_reads/1e6) %>%
  summarize(variance= var(dataset_value),
            min = min(dataset_value),
            max = max(dataset_value),
            median = median(dataset_value),
            p25 = quantile(dataset_value, 0.25),
            p75 = quantile(dataset_value, 0.75),
            iqr = IQR(dataset_value))

kable(Total_read_counts %>%
        select(-variance, -p25, -p75), digits = 1)

min	max	median	iqr
0.2	668	61.2	53.3

total_read_count_histogram <-  ggplot(counts_and_meta) + 
  geom_histogram(aes(Total_reads/1E6), 
                 fill = "lightgrey", color = "black", stat = StatBin2) +
      theme_minimal(base_size = 20) +
    theme(axis.line.x = element_line(color="darkgrey", size = 0.5),
        axis.line.y = element_line(color="darkgrey", size = 0.5)) +
  xlab("Total reads") +
    ylab("Datasets")

total_read_count_histogram

## `stat_bin()` using `bins = 30`. Pick better value with `binwidth`.

Calculate percentages

 read_counts_with_read_type_fractions <- counts_and_meta  %>%
  arrange(desc(Total_reads)) %>%
  mutate(
    pct_unmapped_of_total = (Total_reads - Mapped) / Total_reads,
    pct_dupe_of_mapped = (Mapped - MND) / Mapped,
    pct_non_exonic_of_non_dupe = (MND - MEND) / MND
  )

read_type_fractions_long <- read_counts_with_read_type_fractions %>%
  pivot_longer(cols = starts_with("pct_"), names_to = "read_type_fraction_name", values_to = "dataset_value")

Summarize read type fractions

comparison_of_distributions <- read_type_fractions_long %>% 
  group_by(read_type_fraction_name) %>%
  summarize(variance= var(dataset_value),
            min = min(dataset_value),
            max = max(dataset_value),
            median = median(dataset_value),
            p25 = quantile(dataset_value, 0.25),
            p75 = quantile(dataset_value, 0.75),
            iqr = IQR(dataset_value))

kable(comparison_of_distributions %>%
      mutate_if(is.double, ~100*.), digits = 3) %>% 
  kable_styling(bootstrap_options = "striped", full_width = T)

read_type_fraction_name	variance	min	max	median	p25	p75	iqr
pct_dupe_of_mapped	5.884	2.853	99.997	26.887	13.450	43.033	29.583
pct_non_exonic_of_non_dupe	4.094	4.495	97.000	24.769	16.189	37.277	21.088
pct_unmapped_of_total	0.605	0.710	76.677	3.414	2.740	6.006	3.266

kable(comparison_of_distributions %>%
        select(-variance, -p25, -p75) %>%
      mutate_if(is.double, ~100*.), digits = 0) %>% 
  kable_styling(bootstrap_options = "striped", full_width = T)

read_type_fraction_name	min	max	median	iqr
pct_dupe_of_mapped	3	100	27	30
pct_non_exonic_of_non_dupe	4	97	25	21
pct_unmapped_of_total	1	77	3	3

statements about read type fractions

See comparison_of_distributions table above

unmapped

# in 77 datasets, more than 25% of reads are unmapped
table(read_counts_with_read_type_fractions$pct_unmapped_of_total>0.25)

## 
## FALSE  TRUE 
##  2102    77

duplicate

# 426 datasets have more than 50% duplicates (Fig 2A).
read_counts_with_read_type_fractions %>%
  mutate(above_50 = pct_dupe_of_mapped>0.50) %>%
  tabyl(above_50)

##  above_50    n   percent
##     FALSE 1753 0.8044975
##      TRUE  426 0.1955025

fiftypct <- read_counts_with_read_type_fractions %>%
  group_by(cohort_code) %>%
  summarize(has_a_dataset_with_more_than_50pct_dupes = any(pct_dupe_of_mapped>0.50),
            median_pct_dupe_of_mapped = median(pct_dupe_of_mapped)) %>%
  mutate(median_lt_50 = median_pct_dupe_of_mapped < 0.5,
         median_lt_50_and_has_a_dataset = median_lt_50 & has_a_dataset_with_more_than_50pct_dupes)


fiftypct %>% tabyl(median_lt_50)

##  median_lt_50  n   percent
##         FALSE  7 0.1458333
##          TRUE 41 0.8541667

fiftypct %>% tabyl(has_a_dataset_with_more_than_50pct_dupes)

##  has_a_dataset_with_more_than_50pct_dupes  n percent
##                                     FALSE 15  0.3125
##                                      TRUE 33  0.6875

fiftypct %>% tabyl(median_lt_50_and_has_a_dataset)

##  median_lt_50_and_has_a_dataset  n   percent
##                           FALSE 22 0.4583333
##                            TRUE 26 0.5416667

exonic

read_counts_with_read_type_fractions %>%
  mutate(above_50 = pct_non_exonic_of_non_dupe>0.50) %>%
  tabyl(above_50)

##  above_50    n   percent
##     FALSE 1849 0.8485544
##      TRUE  330 0.1514456

fiftypcte <- read_counts_with_read_type_fractions %>%
  group_by(cohort_code) %>%
  summarize(has_a_dataset_with_more_than_50pct_ne = any(pct_non_exonic_of_non_dupe>0.50),
            median_pct_dupe_of_mapped = median(pct_non_exonic_of_non_dupe)) %>%
  mutate(median_lt_50 = median_pct_dupe_of_mapped < 0.5,
         median_lt_50_and_has_a_dataset = median_lt_50 & has_a_dataset_with_more_than_50pct_ne)


fiftypcte %>% tabyl(median_lt_50)

##  median_lt_50  n percent
##         FALSE  3  0.0625
##          TRUE 45  0.9375

fiftypcte %>% tabyl(has_a_dataset_with_more_than_50pct_ne)

##  has_a_dataset_with_more_than_50pct_ne  n percent
##                                  FALSE 36    0.75
##                                   TRUE 12    0.25

fiftypcte %>% tabyl(median_lt_50_and_has_a_dataset)

##  median_lt_50_and_has_a_dataset  n percent
##                           FALSE 39  0.8125
##                            TRUE  9  0.1875

Correlation of depth of sequencing to duplicate read fraction

Does the depth of sequencing correlate to the number of duplicates? How much variance does that explain?

cor_tot_reads_and_dupes <- cor(read_counts_with_read_type_fractions$Total_reads, 
    read_counts_with_read_type_fractions$pct_dupe_of_mapped,
    method = c("pearson"))
round(cor_tot_reads_and_dupes, 2)

## [1] 0.58

round(cor_tot_reads_and_dupes^2, 2)

## [1] 0.34

Figure S1

Plot correlation of depth of sequencing to duplicate read fraction

this_data <- read_counts_with_read_type_fractions %>%
  select(Total_reads, pct_dupe_of_mapped)

## Adding missing grouping variables: `cohort_code`

this_plot_title <- "Correlation of Total_reads and % Duplicates"

these_stats <- this_data  %>%
  ungroup %>%
  summarize(r_corr = round(cor(Total_reads, pct_dupe_of_mapped), 2))

fs1 <- ggplot(this_data,
       aes(x=Total_reads/1E6, y=pct_dupe_of_mapped)) + 
  geom_point(alpha = 0.5, shape = 20, size =0.1) +
  geom_smooth(method = "lm", color = "black", se = FALSE) +
  geom_label(data = these_stats, 
             aes(label=paste0("r=", r_corr)), 
             x=max(this_data$Total_reads/1E6),
             y=max(this_data$pct_dupe_of_mapped),
             hjust = 1, vjust = 1
             ) +
    theme_minimal(base_size = 20) +
    theme(axis.line.x = element_line(color="darkgrey", size = 0.5),
        axis.line.y = element_line(color="darkgrey", size = 0.5)) +
  ggtitle(this_plot_title)  + 
  theme(legend.position="none", plot.title=element_text(size=22)) +
  xlab("Read counts (million)") +
  ylab("% Duplicates")


ggsave("results/Figure_S1.png", fs1 + ggtitle(""))

## Saving 7 x 5 in image

## `geom_smooth()` using formula 'y ~ x'

ggsave("results/Figure_S1.pdf", fs1 + ggtitle(""))

## Saving 7 x 5 in image
## `geom_smooth()` using formula 'y ~ x'

Generate schematic defining read types

colors_for_read_types <- these_colors_with_MEND
names(colors_for_read_types) <- gsub(" reads", "", names(these_colors_with_MEND))

colors_for_read_types <- c(colors_for_read_types, 
                           "Genome" = "darkblue",
                           "Exon" = "darkblue",
                           "Mapped reads" = "darkgrey")

plot1_colnames <- c("label", "x1", "x2", "y1", "y2")

read_type_schematic_data_as_vector <- c("Duplicate", 3, 4, 5, 6, 
                       "MEND", 3, 4, 4, 5, 
                       "MEND", 2.5, 3.5, 3, 4, 
                       "Non-exonic", 4.5, 5.5, 3, 4,
                       "Unmapped", 6, 7, 3, 4,
                       "Exon", 2.5, 4, 1, 2,
                       "Mapped reads", 2.8, 2.8, 6, 7)

rows_in_schematic <- length(read_type_schematic_data_as_vector)/length(plot1_colnames)

read_type_schematic_data <- matrix(read_type_schematic_data_as_vector, 
                     ncol = length(plot1_colnames), byrow = TRUE,
                     dimnames = list(c(1:rows_in_schematic), plot1_colnames)) %>%
  as_tibble %>%
  type_convert() %>%
  mutate(
    base_color = colors_for_read_types[match(label, names(colors_for_read_types))],
    border_color = case_when(
      label %in% c("Mapped reads", "Genome", "MEND", "Exon") ~ NA_character_,
      TRUE ~ base_color),
    fill_color = ifelse(label %in% c("MEND", "Exon"),  base_color, "white"),
    text_color = case_when(
      label %in% c("MEND", "Exon")~  "white",
      TRUE ~ base_color),
    mid_bar_x = map2_dbl(x1, x2,  function(x, y) mean(c(x,y))),
    mid_bar_y = map2_dbl(y1, y2,  function(x, y) mean(c(x,y)))
  )

## Parsed with column specification:
## cols(
##   label = col_character(),
##   x1 = col_double(),
##   x2 = col_double(),
##   y1 = col_double(),
##   y2 = col_double()
## )

padding_size = 0.35
end_of_box <- sort(read_type_schematic_data$x2,partial=length(read_type_schematic_data$x2)-1)[length(read_type_schematic_data$x2)-1] + padding_size
read_type_schematic <- ggplot(read_type_schematic_data, 
                              aes(xmin=x1, xmax=x2, ymin=y1, ymax = y2, 
                                  fill = fill_color, color = border_color)) +
  geom_rect() +
  
  geom_segment(x=min(read_type_schematic_data$x1-padding_size), y=1.5, xend = 5.75, yend = 1.5, color = "darkblue") +   
  geom_text(aes(label = label, x = mid_bar_x, y=mid_bar_y,
                color = text_color), 
            size = 4,
            fontface = "bold") +
  geom_rect(aes(xmin = min(x1 - padding_size), 
                ymin = 2.5, 
                xmax = end_of_box,
                ymax=max(y2 + padding_size)), 
            fill = NA, color = "darkgrey", size = 0.25) +
  scale_fill_identity() +
  scale_color_identity() +
  theme_void() + 
  theme(
    panel.grid.major.x = element_line(color="lightgrey", size=0.2),
    ) + 
 scale_x_continuous(
   breaks = seq(min(read_type_schematic_data$x1) - padding_size + 0.25, end_of_box , 0.25))

read_type_schematic

Generate schematic defining read type fractions

stat_names <- c("pct_unmapped_of_total",  "pct_dupe_of_mapped", "pct_non_exonic_of_non_dupe")
stat_labels <- c("Unmapped",  "Duplicate", "Non-exonic") 
complement_stat_names <- c("Mapped reads",  "Non-duplicate reads", "Exonic reads") 
divisor_name = c("total", "mapped", "non-duplicate")

comparison_of_remaining <- read_type_fractions_long %>% 
  mutate(read_type_fraction_name = complement_stat_names[match(read_type_fraction_name, stat_names)]) %>%
  group_by(read_type_fraction_name) %>%
  summarize(min = round(1-max(dataset_value), 2),
    max = round(1-min(dataset_value), 2),
    median = round(1-median(dataset_value), 2),
    p25 = round(1-quantile(dataset_value, 0.25), 2),
    p75 = round(1-quantile(dataset_value, 0.75), 2)) %>%
#      mutate(bar_label = paste0 (read_type_fraction_name, "\n(", 100*min, "-", 100*max, "% of ", divisor_name[match(read_type_fraction_name, complement_stat_names)], "; x͂=",100* median, "%)"))
mutate(bar_label = paste0 (read_type_fraction_name, "\n(", 100*min, "-", 100*max, "% of ", divisor_name[match(read_type_fraction_name, complement_stat_names)], "; med.=", 100* median, "%)"))

positive_bars <- comparison_of_remaining %>%
  select(-min, -max, -p25, -p75) %>%
  mutate(read_type_fraction_label = read_type_fraction_name, 
         position = "1") %>%
  rename(abs_median = median)

negative_bars <- tibble(read_type_fraction_name = positive_bars$read_type_fraction_name,
                        read_type_fraction_label = stat_labels[match(read_type_fraction_name, complement_stat_names)],
                        bar_label = read_type_fraction_label,
                        abs_median = 1-positive_bars$abs_median,
                        position = "2")

total_bar <- tibble(read_type_fraction_name = "Total reads",
                    read_type_fraction_label = read_type_fraction_name,
                    bar_label = "Total reads\n(100% of reads)",
                    abs_median = 1,
                    position = "1"
                    )


MEND_stats_of_total <- counts_and_meta  %>%
  arrange(desc(Total_reads)) %>%
  mutate(
    pct_MEND_of_total = (Total_reads - MEND) / Total_reads
  ) %>%
  ungroup %>% 
  summarize(min = round(1-max(pct_MEND_of_total), 2),
    max = round(1-min(pct_MEND_of_total), 2),
    median = round(1-median(pct_MEND_of_total), 2))

MEND_bar <- tibble(read_type_fraction_name = "MEND reads",
                   read_type_fraction_label = read_type_fraction_name,
                   bar_label = with(MEND_stats_of_total, paste0 ("MEND reads\n(", 100*min, "-", 100*max, "% of total reads; med.=",100* median, "%)")),
                   abs_median = 1,
                   position = "1"
)

bar_names_in_order <- c("Total reads", "Mapped reads", "Non-duplicate reads", "Exonic reads", "MEND reads")
  
all_bars <- bind_rows(positive_bars, negative_bars, total_bar, MEND_bar) %>%
  mutate(read_type_fraction_name = factor(read_type_fraction_name, 
                                          levels = bar_names_in_order))

cat_space <- all_bars %>% filter(position == 1) %>%
  arrange(read_type_fraction_name) %>%
  select(read_type_fraction_name, abs_median) %>%
  ungroup %>%
  mutate(lagged1_abs_median = lag(abs_median, default =1),
         lagged2_abs_median = lag(abs_median, n=2, default =1),
         category_space = lagged1_abs_median*lagged2_abs_median
         ) %>%
  select(read_type_fraction_name, category_space)

these_colors_with_MEND = c(these_colors, "MEND reads"="#000000")


all_bars_rel <- left_join(all_bars, cat_space, by = "read_type_fraction_name") %>%
  mutate(rel_median=ifelse(read_type_fraction_name == "MEND reads",
                           abs_median[read_type_fraction_label=="Exonic reads"]*
                             category_space[read_type_fraction_label=="Exonic reads"], 
                           abs_median*category_space),
         read_type_fraction_name = factor(read_type_fraction_name, levels = rev(bar_names_in_order)),
         position = factor(position, levels = rev(sort(unique(position)))),
         bar_color = these_colors_with_MEND[match(read_type_fraction_name, names(these_colors_with_MEND))],
         fill_val = ifelse(position == 1, bar_color, NA),
         border_color_val = bar_color,
         text_color = ifelse(position ==1, "white", "black"),
font_size = ifelse(abs_median<0.1, 3,3.5),
text_angle = ifelse(abs_median<0.1, 90,0)
         )

read_type_fractions_schematic

read_type_fractions_schematic <- ggplot(all_bars_rel, aes(y=read_type_fraction_name,
                x=rel_median, group = position)) + 
   geom_col(aes(fill = fill_val,
                color = border_color_val
                )) +
   geom_text(aes(label=bar_label,
                 color = text_color,
                 size = font_size,
                 angle= text_angle), 
             position = position_stack(vjust = .5),
              fontface = "bold") +
   scale_fill_identity() +
   scale_color_identity()  +
  scale_size_identity() + 
   theme_void() +
  theme(axis.title.y=element_blank(),
        axis.text.y=element_blank(),
        axis.ticks.y=element_blank()) 


ggplot(all_bars_rel, aes(y=read_type_fraction_name,
                x=rel_median, group = position)) + 
   geom_col(aes(fill = fill_val,
                color = border_color_val
                )) +
   geom_text(aes(label=read_type_fraction_label,
                 color = text_color,
                 size = font_size,
                 angle = text_angle), 
             position = position_stack(vjust = .5),
              fontface = "bold") +
   scale_fill_identity() +
   scale_color_identity()  +
  scale_size_identity() + 
   theme_void() +
  theme(axis.title.y=element_blank(),
        axis.text.y=element_blank(),
        axis.ticks.y=element_blank())

read_type_fractions_schematic

Figure 2

figure_name <- "f2"

f2 <- plot_grid(read_type_schematic,
read_type_fractions_schematic,
nrow=2,
rel_heights = c(1, 2),
labels = c("A", "B")) +
   draw_label(paste(figure_name, Sys.time()),
              x = 0, y = 0.01, hjust = 0, size = 6) 
# 
# f2 <- plot_grid(left_side,
# right_side,
# ncol = 2) +
#    draw_label(paste(figure_name, Sys.time()),
#               x = 0, y = 0.02, hjust = 0, size = 6) 

f2

ggsave("results/Figure_2.png", f2, height=6, width=5)
ggsave("results/Figure_2.pdf", f2, height=6, width=5)

stat_names <- c("pct_unmapped_of_total",  "pct_dupe_of_mapped", "pct_non_exonic_of_non_dupe")
stat_label <- c("% unmapped", "% duplicate \n(of mapped)", "% non-exonic \n(of non-duplicate)")
names(stat_label) <- stat_names

Plot read fractions

colors_for_stat_names <- these_colors[c("Mapped reads", "Non-duplicate reads","Exonic reads")]
names(colors_for_stat_names) <- stat_names

these_colors <- c("#A6CEE3", "#1F78B4", "#B2DF8A", "#33A02C", "#FB9A99", "#E31A1C", 
"#FDBF6F", "#FF7F00")
# light blue, dark blue, etc: green, red, orange (then purple and brown)
names(these_colors) <-  c("not assigned", "Total reads", "Non-exonic reads",  "Exonic reads", "Duplicate reads",  "Non-duplicate reads",  "Unmapped reads", "Mapped reads")

# 6 x 11 was a good ratio, but the text was too small

read_type_fractions_long_for_histogram <- read_type_fractions_long %>%
  mutate(read_type_fraction_name = factor(read_type_fraction_name,
                                          levels = stat_names))

read_type_fractions_histogram <-  ggplot(read_type_fractions_long_for_histogram) +
    geom_histogram(aes(x = dataset_value, color = read_type_fraction_name), 
                   fill = "white", stat = StatBin2)  +
  scale_color_manual(values = colors_for_stat_names) +
  facet_wrap(~read_type_fraction_name, 
             nrow = 1, 
             scales = "free_y",
             labeller = labeller(
               read_type_fraction_name = stat_label
               )
             ) +
  ylab("Datasets") +
  xlab(paste0("Read type percentages in ", length(unique(read_type_fractions_long_for_histogram$dataset_id)),  " datasets")) +
  scale_x_continuous(breaks = seq(0, 1, by = 0.5)) +
    theme_minimal() +
    theme(axis.line.x = element_line(color="darkgrey", size = 0.5),
        axis.line.y = element_line(color="darkgrey", size = 0.5)) +
    theme(strip.text.x = element_text(size = 12, face = "bold")) +
    theme(legend.position = "none")

read_type_fractions_histogram

## `stat_bin()` using `bins = 30`. Pick better value with `binwidth`.

Calculate MEND reads as a fraction of total

read_counts_with_MEND_fractions <- counts_and_meta  %>%
  arrange(desc(Total_reads)) %>%
  mutate(pct_MEND_of_total = MEND /Total_reads)

ggplot(read_counts_with_MEND_fractions) + 
  geom_histogram(aes(pct_MEND_of_total), stat = StatBin2) +
    theme_minimal(base_size = 20) +
    theme(axis.line.x = element_line(color="darkgrey", size = 0.5),
        axis.line.y = element_line(color="darkgrey", size = 0.5))

## `stat_bin()` using `bins = 30`. Pick better value with `binwidth`.

MEND_pct <- read_counts_with_MEND_fractions %>% 
  ungroup %>%
  mutate(dataset_value = pct_MEND_of_total) %>%
  summarize(variance= var(dataset_value),
            min = min(dataset_value),
            max = max(dataset_value),
            median = median(dataset_value),
            p25 = quantile(dataset_value, 0.25),
            p75 = quantile(dataset_value, 0.75),
            iqr = IQR(dataset_value))

kable(MEND_pct %>%
        select(-variance, -p25, -p75) %>%
      mutate_if(is.double, ~100*.), digits = 0) %>% 
  kable_styling(bootstrap_options = "striped", full_width = F)

min	max	median	iqr
0	79	50	31

head(sort(read_counts_with_MEND_fractions$pct_MEND_of_total))

## [1] 0.0000267009 0.0002533711 0.0003651723 0.0003767257 0.0003997294
## [6] 0.0004279584

sum(read_counts_with_MEND_fractions$pct_MEND_of_total<0.25)

## [1] 355

read_counts_with_MEND_fractions %>%
  ungroup %>%
  mutate(pct_under_25 = pct_MEND_of_total<0.25) %>%
  tabyl(pct_under_25) %>%
  adorn_totals()

##  pct_under_25    n   percent
##         FALSE 1824 0.8370812
##          TRUE  355 0.1629188
##         Total 2179 1.0000000

read_counts_with_MEND_fractions %>%
  ungroup %>%
  filter(Mapped > 30E6) %>%
  mutate(pct_under_25 = pct_MEND_of_total<0.25) %>%
  tabyl(pct_under_25) %>%
  adorn_totals()

##  pct_under_25    n   percent
##         FALSE 1739 0.8368624
##          TRUE  339 0.1631376
##         Total 2078 1.0000000

Show per-cohort distribution of read_type_fractions

figure_name <- "fracs_by_group"

read_type_fractions_long_for_boxplot <- read_type_fractions_long_for_histogram %>%
  ungroup %>%
  mutate(boxplot_label = fct_reorder(paste0(as.character(cohort_code), ", n=", n_in_cohort), n_in_cohort))


read_type_fractions_per_cohort_boxplot <- ggplot(read_type_fractions_long_for_boxplot) +
  geom_boxplot(aes(x = dataset_value, y=boxplot_label), outlier.size = 0.5)  +
  facet_wrap(~read_type_fraction_name, 
             nrow = 1, 
             labeller = labeller(
               read_type_fraction_name = stat_label
             )
  ) +
  ylab("Cohorts") +
  xlab(paste0("Read type percentages in ", length(unique(read_type_fractions_long_for_boxplot$dataset_id)),  " datasets")) +
  scale_x_continuous(breaks = seq(0, 1, by = 0.5)) +
    theme_minimal() +
    theme(axis.line.x = element_line(color="darkgrey", size = 0.5),
        axis.line.y = element_line(color="darkgrey", size = 0.5)) +
  theme(strip.text.x = element_text(size = 12, face = "bold")) +
  theme(legend.position = "none")


read_type_fractions_per_cohort_boxplot

Characterize Cohort 4

read_type_names <- c("Total_reads", "Mapped",  "MND", "MEND")

this_cohort_name <- "EGAD00001003279"

this_cohort_code <- subset(read_counts_with_read_type_fractions, cohort_name == this_cohort_name)$cohort_code[1]

this_cohort_code

## [1] "Cohort_4"

in MS

 read_counts_with_read_type_fractions %>%
  filter(cohort_code == this_cohort_code) %>%
   mutate(gt_98 = pct_dupe_of_mapped > 0.98) %>%
   tabyl(gt_98) %>%
   add_totals_row

## Warning: 'add_totals_row' is deprecated.
## Use 'adorn_totals("row")' instead.
## See help("Deprecated")

##  gt_98   n   percent
##  FALSE  55 0.4330709
##   TRUE  72 0.5669291
##  Total 127 1.0000000

read_counts_with_read_type_fractions %>%
  filter(cohort_code == this_cohort_code) %>%
  filter(pct_dupe_of_mapped > 0.98) %>%
  pull(Total_reads) %>% min

## [1] 178294341

other

 counts_and_meta_long <- counts_and_meta  %>%
  pivot_longer(cols = c(Total_reads, Mapped, MEND, MND), names_to = "read_type_name", values_to = "dataset_value")  %>%
   ungroup %>%
   mutate(read_type_name = factor(read_type_name, levels = read_type_names))

counts_and_meta_long_one_proj <-  counts_and_meta_long %>%
  filter(cohort_code == this_cohort_code)

table(counts_and_meta_long_one_proj$read_type_name)

## 
## Total_reads      Mapped         MND        MEND 
##         127         127         127         127

# 78 datasets in the cohort have fewer than 0.2 million MEND reads. 
sum((counts_and_meta_long_one_proj %>% filter(read_type_name=="MEND"))$dataset_value < 200000)

## [1] 78

those_datasets <- counts_and_meta_long_one_proj %>% filter(read_type_name=="MEND", dataset_value < 200000) %>% pull(dataset_id)


summary((counts_and_meta_long_one_proj %>% filter(read_type_name=="Total_reads", dataset_id %in% those_datasets))$dataset_value)

##      Min.   1st Qu.    Median      Mean   3rd Qu.      Max. 
## 178294341 199916664 208667776 211606891 223832191 247618832

max_plots_to_include <- 10
n_datasets_to_plot <- ifelse(n_distinct(counts_and_meta_long_one_proj$dataset_id)>max_plots_to_include, 
                         max_plots_to_include, 
                         n_distinct(counts_and_meta_long_one_proj$dataset_id))
plot_word <- ifelse(n_datasets_to_plot==max_plots_to_include, max_plots_to_include, paste("all", n_distinct(counts_and_meta_long_one_proj$dataset_id)))

set.seed(100)
datasets_to_plot <- sample(unique(counts_and_meta_long_one_proj$dataset_id), n_datasets_to_plot)

ggplot(subset(counts_and_meta_long_one_proj, dataset_id %in% datasets_to_plot)) + 
  geom_col(aes(x=read_type_name, y=dataset_value/1e6, fill = read_type_name)) +
  facet_wrap(~dataset_id, ncol = 1, strip.position="right")  +
    theme_minimal(base_size = 20) +
    theme(axis.line.x = element_line(color="darkgrey", size = 0.5),
        axis.line.y = element_line(color="darkgrey", size = 0.5)) +
  theme(strip.text.y = element_text(angle = 0)) +
  scale_fill_brewer(palette = "Set1")  +
  theme(axis.text.x = element_text(angle = 90, hjust = 1, vjust = 0.5)) +
  ggtitle(paste("Read counts for", plot_word, "datasets from", this_cohort_code)) +
  coord_flip()

ggplot(counts_and_meta_long_one_proj %>% filter(read_type_name == "MEND")) + 
  geom_histogram(aes(dataset_value/1e6), stat = StatBin2) +
    theme_minimal(base_size = 20) +
    theme(axis.line.x = element_line(color="darkgrey", size = 0.5),
        axis.line.y = element_line(color="darkgrey", size = 0.5)) +
  ggtitle(paste("Distribution of MEND counts in", this_cohort_code))

## `stat_bin()` using `bins = 30`. Pick better value with `binwidth`.

bl <- colorRampPalette(c("navy","royalblue","lightskyblue"))(200)                      
re <- colorRampPalette(c("mistyrose", "red2","darkred"))(200)


ggplot(counts_and_meta %>% 
         filter(cohort_code == this_cohort_code) %>%
         mutate(pct_dupes = (Mapped - MND)/Mapped)) + 
  geom_point(aes(x=MEND/1e6, y=pct_dupes, fill = Total_reads/1e6), shape=21, color = "black") +
    theme_minimal(base_size = 20) +
    theme(axis.line.x = element_line(color="darkgrey", size = 0.5),
        axis.line.y = element_line(color="darkgrey", size = 0.5)) +
  scale_fill_gradientn(colours = c(bl,"white", re)) +
  ggtitle(paste("Relationship of dupe fraction to MEND count in", this_cohort_code), "For detecting whether datasets with less info were sequenced more deeply. \nNormally, higher depth datasets have more MEND reads and more duplicate \nreads than the same dataset sequenced at a lower total depth.")

counts_and_meta_long_one_proj %>%
  select(seq_length, dataset_id) %>%
  distinct %>%
  tabyl(seq_length)

##  seq_length   n percent
##          51 127       1

Expressed genes

What is the distribution of measured genes?

gene_counts_long <- counts_and_meta %>%
  pivot_longer(cols = starts_with("genes_"), names_to = "Expression_threshold")  %>%
  mutate(Expression_threshold = paste(str_replace(Expression_threshold, "genes_expressed_above_", "genes > "), "TPM"))  


ggplot(gene_counts_long) + 
  geom_histogram(aes(x=value/1e3, fill = Expression_threshold), stat = StatBin) +
  facet_wrap(~Expression_threshold)  +
    theme_minimal() +
  scale_fill_brewer(palette = "Set1") +
    theme(axis.line.x = element_line(color="darkgrey", size = 0.5),
        axis.line.y = element_line(color="darkgrey", size = 0.5)) +
    theme(legend.position = "none") +
  ylab("Datasets") +
  xlab("Measured genes")

## `stat_bin()` using `bins = 30`. Pick better value with `binwidth`.

What are the exact lowest gene counts?

sort(counts_and_meta$genes_expressed_above_0) [1:100]

##   [1]     9     9    12    12    13    14    14    15    16    16    17
##  [12]    18    18    18    18    18    18    19    19    19    19    20
##  [23]    20    20    20    20    20    21    21    21    21    21    21
##  [34]    22    22    22    23    23    23    23    23    24    24    24
##  [45]    24    24    24    24    25    25    25    26    26    26    26
##  [56]    27    27    27    27    28    28    28    28    29    29    29
##  [67]    29    29    30    30    31    31    32    33    35    36    37
##  [78]    40  6098  7107 11830 15323 16793 16837 16943 17233 17531 18288
##  [89] 18588 18704 19626 19693 19749 19781 19942 19963 20063 20120 20203
## [100] 20247

correlations with all datasets

# library(corrr)

counts_and_meta %>% 
    ungroup %>% 
    select_if(is.double) %>%
       correlate() %>%
  filter(rowname %in% read_type_names) %>%
  rename(Read_type_count=rowname) %>% 
  mutate(Read_type_count = factor(Read_type_count, levels = read_type_names)) %>%
  arrange(Read_type_count) %>%
  select(c(Read_type_count, starts_with("genes_expressed"))) %>%
  kable(caption = "Correlations with all datasets", digits = 2) %>% 
  kable_styling(bootstrap_options = "striped", full_width = F)

## 
## Correlation method: 'pearson'
## Missing treated using: 'pairwise.complete.obs'

Correlations with all datasets
Read_type_count	genes_expressed_above_0	genes_expressed_above_1	genes_expressed_above_2	genes_expressed_above_3	genes_expressed_above_4	genes_expressed_above_5
Total_reads	-0.20	-0.40	-0.40	-0.40	-0.39	-0.39
Mapped	-0.19	-0.41	-0.41	-0.41	-0.40	-0.39
MND	0.51	0.24	0.20	0.18	0.17	0.16
MEND	0.48	0.27	0.26	0.26	0.26	0.26

correlations with all but low gene count datasets

counts_and_meta %>% 
  filter(genes_expressed_above_0 > min_genes_gt_0) %>%
    ungroup %>% 
    select_if(is.double) %>%
       correlate() %>%
  filter(rowname %in% read_type_names) %>%
  rename(Read_type_count=rowname) %>% 
  mutate(Read_type_count = factor(Read_type_count, levels = read_type_names)) %>%
  arrange(Read_type_count) %>%
  select(c(Read_type_count, starts_with("genes_expressed"))) %>%
  kable(caption = "Correlations with all but low gene count datasets", digits = 2) %>% 
  kable_styling(bootstrap_options = "striped", full_width = F)

## 
## Correlation method: 'pearson'
## Missing treated using: 'pairwise.complete.obs'

Correlations with all but low gene count datasets
Read_type_count	genes_expressed_above_0	genes_expressed_above_1	genes_expressed_above_2	genes_expressed_above_3	genes_expressed_above_4	genes_expressed_above_5
Total_reads	0.13	-0.26	-0.28	-0.28	-0.28	-0.28
Mapped	0.21	-0.25	-0.26	-0.27	-0.27	-0.27
MND	0.51	0.04	0.02	0.00	0.00	0.00
MEND	0.44	0.08	0.09	0.11	0.11	0.12

Plot correlations betweeen gene counts and read types

Groom data

expr_gene_and_read_counts_to_plot <- counts_and_meta %>%
  pivot_longer (cols = c(MND, MEND, Total_reads, Mapped), names_to = "Read_type", values_to = "Read_count") %>%
  pivot_longer (cols = starts_with("genes_expressed"), names_to = "Expression_threshold", values_to = "Gene_count") %>%
  mutate(Read_type = factor(Read_type, levels = read_type_names))

Identify datasets with unusual gene counts or read depths

datasets_with_normal_expressed_gene_numbers <- counts_and_meta %>%
  filter(genes_expressed_above_0 > min_genes_gt_0) %>%
  pull(dataset_id)

datasets_with_outlier_gene_counts <- counts_and_meta %>%
  ungroup %>%
  filter(is_outlier(genes_expressed_above_0)) %>%
  pull(dataset_id) 

datasets_with_outlier_depths <- counts_and_meta %>%
  ungroup %>%
  filter(is_outlier(Total_reads)) %>%
  pull(dataset_id)

Function for calculating gene count correlations and plotting results

plot_gene_count_correlations <- function(this_data = expr_gene_and_read_counts_to_plot, this_plot_title = "gene count correlations"){

this_data <- this_data %>% 
  mutate(Expression_threshold = paste(str_replace(Expression_threshold, "genes_expressed_above_", "genes > "), "TPM"))  
  
  
these_stats <- this_data  %>%
group_by(Read_type, Expression_threshold) %>%
  summarize(r_corr = round(cor(Gene_count, Read_count), 2)) 

# unique(this_data$Read_type)
# dput(unique(this_data$Read_type))
# these_colors_with_MEND

colors_for_correlation_plot <- c("Total_reads"="#1F78B4", "Mapped"="#FF7F00", 
"MND"="#E31A1C", "MEND"="#000000")

ggplot(this_data,
       aes(x=Read_count/1E6, y=Gene_count/1e3, color = Read_type)) + 
  geom_point(alpha = 0.5, shape = 20, size =0.1) +
  geom_smooth(method = "lm", color = "black") +
  geom_label(data = these_stats, 
             aes(label=paste0("r=", r_corr)), 
             x=max(this_data$Read_count/1E6),
             y=max(this_data$Gene_count/1e3),
             hjust = 1, vjust = 1
             ) +
    theme_minimal() +
    theme(axis.line.x = element_line(color="darkgrey", size = 0.5),
        axis.line.y = element_line(color="darkgrey", size = 0.5)) +
  scale_color_manual(values = colors_for_correlation_plot) +
  facet_grid(Read_type~Expression_threshold) +
  ggtitle(this_plot_title)  + 
  theme(legend.position="none") +
  xlab("Read counts (million)") +
  ylab("Gene counts (thousand)")

}

Figure S2

fs2 <- plot_gene_count_correlations(expr_gene_and_read_counts_to_plot,
                             "Correlations calculated across all datasets")

fs2

## `geom_smooth()` using formula 'y ~ x'

ggsave("results/Figure_S2.png", fs2 + ggtitle(""))

## Saving 7 x 5 in image
## `geom_smooth()` using formula 'y ~ x'

ggsave("results/Figure_S2.pdf", fs2 + ggtitle(""))

## Saving 7 x 5 in image
## `geom_smooth()` using formula 'y ~ x'

for all datasets

plot_gene_count_correlations(expr_gene_and_read_counts_to_plot %>% 
  filter(dataset_id %in% datasets_with_normal_expressed_gene_numbers),
  paste("Correlations in datasets with more than", min_genes_gt_0, "genes expressed above 0"))

## `geom_smooth()` using formula 'y ~ x'

plot_gene_count_correlations(expr_gene_and_read_counts_to_plot %>% 
  filter(! dataset_id %in% datasets_with_outlier_gene_counts),
  paste("Correlations calculated across all but outlier gene count datasets"))

## `geom_smooth()` using formula 'y ~ x'

plot_gene_count_correlations(expr_gene_and_read_counts_to_plot %>% 
  filter(! dataset_id %in% datasets_with_outlier_depths),
  paste("Correlations calculated across all but outlier read depth datasets"))

## `geom_smooth()` using formula 'y ~ x'

plot_gene_count_correlations(expr_gene_and_read_counts_to_plot %>% 
  filter(! dataset_id %in% datasets_with_outlier_depths, 
         ! dataset_id %in% datasets_with_outlier_gene_counts
         ),
  paste("Correlations calculated across all but datasets with outlier read depth or gene count"))

## `geom_smooth()` using formula 'y ~ x'

plot_gene_count_correlations(
  expr_gene_and_read_counts_to_plot %>% 
    filter(disease ==  "acute lymphoblastic leukemia"),
  "Correlations calculated among acute lymphoblastic leukemia datasets")

## `geom_smooth()` using formula 'y ~ x'

corr plot for figure

max_total_read_depth <- 250*1e6
print(min_genes_gt_0)

## [1] 100

sum(counts_and_meta$genes_expressed_above_0 <= min_genes_gt_0)

## [1] 78

sum(counts_and_meta$Total_reads > max_total_read_depth)

## [1] 105

# min_genes_gt_0
exclude_for_depth <- counts_and_meta %>%
  filter(Total_reads > max_total_read_depth) %>%
  pull(dataset_id)

exclude_for_gene_count <- counts_and_meta %>%
  filter(genes_expressed_above_0 < min_genes_gt_0) %>%
  pull(dataset_id)


this_data <- expr_gene_and_read_counts_to_plot %>% 
      filter(
        Expression_threshold %in% paste0("genes_expressed_above_", 1:3),
       ! dataset_id %in% exclude_for_depth, 
        ! dataset_id %in% exclude_for_gene_count
      ) %>%
  mutate(Expression_threshold = paste(str_replace(Expression_threshold, "genes_expressed_above_", "genes > "), "TPM"))  

n_distinct(this_data$dataset_id)

## [1] 1996

this_plot_title <- paste("Correlations calculated across all but datasets with excessive read depth or low gene count")

these_stats <- this_data  %>%
group_by(Read_type, Expression_threshold) %>%
  summarize(r_corr = round(cor(Gene_count, Read_count), 2))

colors_for_correlation_plot <- c("Total_reads"="#1F78B4", "Mapped"="#FF7F00", 
"MND"="#E31A1C", "MEND"="#000000")

cor_plots_excluding_high_read_depth_and_low_gene_count_datasets <- ggplot(this_data,
       aes(x=Read_count/1E6, y=Gene_count/1e3, color = Read_type)) + 
  geom_point(alpha = 0.5, shape = 20, size =0.1) +
  geom_smooth(method = "lm", color = "black") +
  geom_label(data = these_stats, 
             aes(label=paste0("r=", r_corr)), 
             x=max(this_data$Read_count/1E6),
             y=max(this_data$Gene_count/1e3),
             hjust = 1, vjust = 1
             ) +
    theme_minimal() +
    theme(axis.line.x = element_line(color="darkgrey", size = 0.5),
        axis.line.y = element_line(color="darkgrey", size = 0.5),
        strip.text = element_text(face = "bold")) +
  scale_color_manual(values = colors_for_correlation_plot) +
  facet_grid(Read_type~Expression_threshold) +
  theme(legend.position="none") +
  scale_x_continuous("Read counts (million)", n.breaks = 4) +
  ylab("Gene counts (thousand)") 


cor_plots_excluding_high_read_depth_and_low_gene_count_datasets

## `geom_smooth()` using formula 'y ~ x'

Figure 3

f3a <- read_type_fractions_histogram +
            theme(strip.text.x = element_text(size = 10, face = "bold"),
                  axis.title.x=element_blank(),
                  axis.text.x=element_blank())

f3b <- read_type_fractions_per_cohort_boxplot +
            theme(strip.text.x = element_blank(),
                  strip.background = element_blank(),
                  strip.text = element_blank())

f3ab <- plot_grid(f3a, 
                  f3b,
                  ncol = 1,
                  rel_heights = c(1.5, 6),
                  align = "v",
                  labels = "AUTO")

## `stat_bin()` using `bins = 30`. Pick better value with `binwidth`.

f3c <- plot_grid(cor_plots_excluding_high_read_depth_and_low_gene_count_datasets,
          labels = "C")

## `geom_smooth()` using formula 'y ~ x'

f3 <- plot_grid(f3ab,
                 f3c,
                 nrow=1,
                 rel_widths = c(4,2.3))

figure_name <- "f3"
f3_labeled <- f3 +   draw_label(paste(figure_name, Sys.time()),
             x = 0, y = 0.02, hjust = 0, size = 6) 


f3_labeled

ggsave("results/Figure_3.png", f3_labeled, height=8, width=10)
ggsave("results/Figure_3.pdf", f3_labeled, height=8, width=10)

Plot sequence length against pct duplicates

ggplot(read_counts_with_read_type_fractions) + 
  geom_point(aes(x=seq_length, y=pct_dupe_of_mapped))

ggplot(read_counts_with_read_type_fractions) + 
  geom_boxplot(aes(x=seq_length, y=pct_dupe_of_mapped, group = seq_length))

Generate bibtex formatted citations for packages

used_pkgs <- c("base", "tidyverse", "janitor", "knitr", "cowplot", "RColorBrewer", "pander", "kableExtra", "snakecase", "corrr")
write_bib(used_pkgs)

## @Manual{R-base,
##   title = {R: A Language and Environment for Statistical Computing},
##   author = {{R Core Team}},
##   organization = {R Foundation for Statistical Computing},
##   address = {Vienna, Austria},
##   year = {2019},
##   url = {https://www.R-project.org/},
## }
## @Manual{R-corrr,
##   title = {corrr: Correlations in R},
##   author = {Edgar Ruiz and Simon Jackson and Jorge Cimentada},
##   year = {2019},
##   note = {R package version 0.4.0},
##   url = {https://CRAN.R-project.org/package=corrr},
## }
## @Manual{R-cowplot,
##   title = {cowplot: Streamlined Plot Theme and Plot Annotations for 'ggplot2'},
##   author = {Claus O. Wilke},
##   year = {2019},
##   note = {R package version 1.0.0},
##   url = {https://CRAN.R-project.org/package=cowplot},
## }
## @Manual{R-janitor,
##   title = {janitor: Simple Tools for Examining and Cleaning Dirty Data},
##   author = {Sam Firke},
##   year = {2019},
##   note = {R package version 1.2.0},
##   url = {https://CRAN.R-project.org/package=janitor},
## }
## @Manual{R-kableExtra,
##   title = {kableExtra: Construct Complex Table with 'kable' and Pipe Syntax},
##   author = {Hao Zhu},
##   year = {2019},
##   note = {R package version 1.1.0},
##   url = {https://CRAN.R-project.org/package=kableExtra},
## }
## @Manual{R-knitr,
##   title = {knitr: A General-Purpose Package for Dynamic Report Generation in R},
##   author = {Yihui Xie},
##   year = {2019},
##   note = {R package version 1.25},
##   url = {https://CRAN.R-project.org/package=knitr},
## }
## @Manual{R-pander,
##   title = {pander: An R 'Pandoc' Writer},
##   author = {Gergely Daróczi and Roman Tsegelskyi},
##   year = {2018},
##   note = {R package version 0.6.3},
##   url = {https://CRAN.R-project.org/package=pander},
## }
## @Manual{R-RColorBrewer,
##   title = {RColorBrewer: ColorBrewer Palettes},
##   author = {Erich Neuwirth},
##   year = {2014},
##   note = {R package version 1.1-2},
##   url = {https://CRAN.R-project.org/package=RColorBrewer},
## }
## @Manual{R-snakecase,
##   title = {snakecase: Convert Strings into any Case},
##   author = {Malte Grosser},
##   year = {2019},
##   note = {R package version 0.11.0},
##   url = {https://CRAN.R-project.org/package=snakecase},
## }
## @Manual{R-tidyverse,
##   title = {tidyverse: Easily Install and Load the 'Tidyverse'},
##   author = {Hadley Wickham},
##   year = {2017},
##   note = {R package version 1.2.1},
##   url = {https://CRAN.R-project.org/package=tidyverse},
## }

Does the depth of sequencing correlate to the number of duplicates? How much variance does that explain?

cor_tot_reads_and_dupes <- cor(read_counts_with_read_type_fractions$Total_reads, 
    read_counts_with_read_type_fractions$pct_dupe_of_mapped,
    method = c("pearson"))
round(cor_tot_reads_and_dupes, 2)

## [1] 0.58

round(cor_tot_reads_and_dupes^2, 2)

## [1] 0.34

SessionInfo

pander(sessionInfo(), compact = FALSE)

R version 3.6.0 (2019-04-26)

Platform: x86_64-apple-darwin15.6.0 (64-bit)

locale: en_US.UTF-8||en_US.UTF-8||en_US.UTF-8||C||en_US.UTF-8||en_US.UTF-8

attached base packages:

stats
graphics
grDevices
utils
datasets
methods
base

other attached packages:

corrr(v.0.4.0)
snakecase(v.0.11.0)
kableExtra(v.1.1.0)
pander(v.0.6.3)
RColorBrewer(v.1.1-2)
cowplot(v.1.0.0)
knitr(v.1.25)
janitor(v.1.2.0)
forcats(v.0.4.0)
stringr(v.1.4.0)
dplyr(v.0.8.5)
purrr(v.0.3.3)
readr(v.1.3.1)
tidyr(v.1.0.0)
tibble(v.3.0.1)
ggplot2(v.3.3.0)
tidyverse(v.1.2.1)

loaded via a namespace (and not attached):

tidyselect(v.1.1.0)
xfun(v.0.10)
lattice(v.0.20-38)
splines(v.3.6.0)
haven(v.2.1.1)
colorspace(v.1.4-1)
vctrs(v.0.3.0)
generics(v.0.0.2)
htmltools(v.0.4.0)
viridisLite(v.0.3.0)
mgcv(v.1.8-29)
yaml(v.2.2.0)
rlang(v.0.4.6)
pillar(v.1.4.3)
glue(v.1.4.1)
withr(v.2.1.2)
modelr(v.0.1.5)
readxl(v.1.3.1)
lifecycle(v.0.2.0)
munsell(v.0.5.0)
gtable(v.0.3.0)
cellranger(v.1.1.0)
rvest(v.0.3.4)
evaluate(v.0.14)
labeling(v.0.3)
fansi(v.0.4.0)
highr(v.0.8)
broom(v.0.7.0)
Rcpp(v.1.0.3)
scales(v.1.1.0)
backports(v.1.1.5)
webshot(v.0.5.1)
jsonlite(v.1.6)
farver(v.2.0.1)
hms(v.0.5.1)
digest(v.0.6.23)
stringi(v.1.4.3)
grid(v.3.6.0)
cli(v.2.0.0)
tools(v.3.6.0)
magrittr(v.1.5)
crayon(v.1.3.4)
pkgconfig(v.2.0.3)
Matrix(v.1.2-17)
ellipsis(v.0.3.0)
xml2(v.1.2.2)
lubridate(v.1.7.4)
assertthat(v.0.2.1)
rmarkdown(v.1.16)
httr(v.1.4.1)
rstudioapi(v.0.10)
R6(v.2.4.1)
nlme(v.3.1-141)
compiler(v.3.6.0)

rna-seq-survey-MS-v20

hbeale

27 August, 2020

Libraries

Config

Settings

Functions

Import data

Colors

Datasets analyzed

Diseases in dataset

Cohorts in dataset

Table S2 - repositories

Table S3 - data access table

Race and gender in dataset

Genders in dataset

Ages in dataset

Read lengths in dataset

Check for counting errors

Summarize total read counts

Calculate percentages

Summarize read type fractions

statements about read type fractions

unmapped

duplicate

exonic

Correlation of depth of sequencing to duplicate read fraction

Does the depth of sequencing correlate to the number of duplicates? How much variance does that explain?

Figure S1

Plot correlation of depth of sequencing to duplicate read fraction

Generate schematic defining read types

Generate schematic defining read type fractions

read_type_fractions_schematic

Figure 2

Plot read fractions

Calculate MEND reads as a fraction of total

Show per-cohort distribution of read_type_fractions

Characterize Cohort 4

in MS

other

Expressed genes

What is the distribution of measured genes?

What are the exact lowest gene counts?

correlations with all datasets

correlations with all but low gene count datasets

Plot correlations betweeen gene counts and read types

Groom data

Identify datasets with unusual gene counts or read depths

Function for calculating gene count correlations and plotting results

Figure S2

for all datasets

corr plot for figure

Figure 3

Plot sequence length against pct duplicates

Generate bibtex formatted citations for packages

Does the depth of sequencing correlate to the number of duplicates? How much variance does that explain?

SessionInfo